The objective of this research project is to develop ex-vivo progenitor expansion techniques using static as well as continuous flow perfusion systems, to explore the future clinical applicability of progenitor cell transfusions in the setting of high-dose chemotherapy for breast cancer. New advances in stem cell isolation technology and the identification of many early-acting synergistic cytokines such as stem cell factor/kit ligand (SCF), interleukin-1 (IL-1), interleukin 11 (IL-11), leukemia inhibitory factor (LIF) and the GM-CSF/IL-3 fusion protein PIXY-321), have made such an endeavor possible. In Aim 1, CD34+ subsets will be characterized in static vs perfusion cultures (CELLMAX), their ability to grow in long-term and short-term colony assays will be correlated with their proliferation and maturation by multiparameter flow cytometry (Project 3). The role of stromal cell contact will be studied with a) SCF expressing Steel lines expressing the membrane bound form of human SCF, and b) with the IL-11, IL-1alpha/beta, CSF producing mesenchymal GCT cell line. In Aim 2 we will examine the efficacy of chemical (4-HC) and immunomagnetic purging methods to remove residual tumor cells from hematopoietic cells undergoing ex-vivo expansion, using a model breast cancer cell line expressing the HSV-TK-Neo gene. Capitalizing on Dr. Leary's expertise in rare cell detection (Project 3), we will quantify breast cancer cells in progenitor cell products derived from patients enrolling in our peripheral stem cell mobilization protocol (Project 1). Lastly, in Aim 3, in collaboration with Drs. Neinhuis and Arceci we will transduce mobilized and/or ex-vivo expanded CD34+ stem cells with retroviral vectors in order to examine the feasibility to expand myeloid progenitor cells retaining high amounts of mdr1 expression. We will capitalize on the available flow cytometric expertise at the University of Rochester, to monitor the frequency of gene transduction into breast cancer cells using these combined tumor cell purging and progenitor expansion protocols. These studies will set the stage for future, more efficient progenitor cell transfusions and efficient stem cell transduction protocols in the therapy of cancer patients and breast cancer in particular.